| 摘要: |
| 目的：运用网络药理学分析黄芪桂枝五物汤治疗三阴性乳腺癌（triple-negative breast cancer，TNBC）的有效活性成分、关键作用靶点和作用机制，并通过体外试验验证其抑制TNBC细胞（MDA-MB-231）的增殖、迁移和集落形成能力。方法：通过中药系统药理数据库（Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform，TCMSP）检索黄芪桂枝五物汤的有效活性成分及其作用靶点，同时利用人类基因数据库（The Human Gene Database，GeneCards）、药物基因学知识库（Pharmacogenetics and Pharmacogenomics Knowledge Base，PharmGkb）和治疗靶点数据库（Therapeutic Target Database，TTD）确定TNBC的相关靶点，并取交集以确定共同作用靶点。将交集靶点导入蛋白质相互作用数据库（Search Tool for the Retrieval of Interacting Genes/Proteins，STRING）平台以构建蛋白质-蛋白质相互作用网络（protein-protein interatction，PPI），并进行基因本体（gene ontology，GO）功能与京都基因和基因组数据库（Kyoto Encyclopedia of Genes and Genomes，KEGG）通路富集分析，使用Cytoscape 3.10.0软件构建网络模型。细胞计数试验盒-8（Cell Counting Kit-8，CCK-8）实验检测黄芪桂枝五物汤提取物（0、20、40、80、120、160、200 μg/mL）对MDA-MB-231细胞增殖活性的影响;细胞划痕实验检测黄芪桂枝五物汤（80 μg/mL）对MDA-MB-231细胞迁移能力的影响;细胞集落实验检测黄芪桂枝五物汤提取物（0、20、40、80 μg/mL）对MDA-MB-231细胞集落形成能力的影响。结果：共筛选出45种活性成分和223个相关作用靶点，TNBC相关靶点共计6 200个，其中191个为交集靶点。PPI网络分析得到核心作用靶点为蛋白激酶B（v-akt murine thymoma viral oncogene homolog 1，AKT1）、白细胞介素-6（interleukin-6，IL-6）、肿瘤抑制蛋白53（tumor protein 53，TP53）、白细胞介素-1B（interleukin-1B，IL-1B）、雌激素受体1（estrogen receptor 1，ESR1）、环氧合酶2（prostaglandin-endoperoxide synthase 2，PTGS2）、半胱氨酸天冬氨酸蛋白酶3（recombinant caspase 3，CASP3）、表皮生长因子受体（epidermal growth factor receptor，EGFR）、原癌基因c-Jun（JUN）、基质金属蛋白酶9（matrix metallopeptidase 9，MMP9）。GO功能主要富集在蛋白质结合、酶结合、蛋白质同源二聚活性等;KEGG通路主要富集在肿瘤相关、脂质与动脉粥样硬化、晚期糖基化终产物（advanced glycation end-products receptor for advanced glycation end-products，AGE-RAGE）信号通路等。CCK-8实验结果显示，黄芪桂枝五物汤提取物（0、20、40、80 μg/mL）可显著抑制MDA-MB-231细胞增殖（P＜0.001），且半数抑制浓度（median inhibitory concentration，IC50）为81.02 μg/mL。细胞划痕实验结果表明，与对照组相比，80 μg/mL的黄芪桂枝五物汤提取物能显著抑制MDA-MB-231细胞迁移能力（P＜0.05）。细胞集落实验结果显示，黄芪桂枝五物汤提取物能抑制MDA-MB-231细胞的集落形成能力（P＜0.05）。结论：黄芪桂枝五物汤通过槲皮素、山柰酚等成分作用于AKT1、TP53等关键靶点，抑制乳腺癌细胞的增殖、迁移和集落形成，本研究揭示了其多靶点、多途径的抗肿瘤机制，为TNBC治疗提供了新的策略。 |
| 关键词: 三阴性乳腺癌 黄芪桂枝五物汤 网络药理学 细胞实验 |
| DOI： |
|
|
| Therapeutic effect of Huangqi Guizhi Wuwu decoction on triple-negative breast cancer：A study based on network pharmacology and cell experiment |
| YE Yangtian,SUN Dao |
| (Hunan Provincial Maternal and Child Health Care Hospital，Changsha 410008，Hunan，China) |
| Abstract: |
| Objective：To investigate the active components，key action targets，and mechanism of action of Huangqi Guizhi Wuwu decoction in the treatment of triple-negative breast cancer (TNBC) based on network pharmacology，and to validate its ability to inhibit the proliferation，migration，and colony formation of TNBC MDA-MB-231 cells through in vitro experiment.Methods：Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform was used to obtain the active components of Huangqi Guizhi Wuwu decoction and their action targets，and the Human Gene Database (GeneCards)，Pharmacogenetics and Pharmacogenomics Knowledge Base，and Therapeutic Target Database were used to determine the targets associated with TNBC;the two groups of targets were intersected to obtain common action targets.The intersecting targets were imported into the Search Tool for the Retrieval of Interacting Genes/Proteins platform to construct a protein-protein interaction (PPI) network，and the gene ontology (GO) functional enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed.Cytoscape 3.10.0 software was used to contruct a network model.Cell Counting Kit-8 (CCK-8) assay was used to observe the effect of the extract of Huangqi Guizhi Wuwu decoction (0，20，40，80，120，160，and 200 μg/mL) on the proliferative activity of MDA-MB-231 cells;wound healing assay was used to observe the effect of Huangqi Guizhi Wuwu decoction (80 μg/mL) on the migration ability of MDA-MB-231 cells;colony formation assay was used to observe the effect of the extract of Huangqi Guizhi Wuwu decoction (0，20，40，and 80 μg/mL) on the colony formation ability of MDA-MB-231 cells.Results：A total of 45 active components and 223 related action targets were obtained，and 6200 targets were obtained for TNBC，among which there were 191 intersecting targets.The PPI network analysis obtained the core action targets of v-akt murine thymoma viral oncogene homolog 1 (AKT1)，interleukin-6，tumor protein 53 (TP53)，interleukin-1B，estrogen receptor 1，prostaglandin-endoperoxide synthase 2，recombinant caspase-3，epidermal growth factor receptor，c-Jun，and matrix metallopeptidase-9.The GO functions were mainly enriched in protein binding，enzyme binding，and protein homodimerization activity，and the KEGG pathways were mainly enriched in cancer-related pathways，lipid and atherosclerosis，and the AGE-RAGE signaling pathway.CCK-8 assay showed that the extract of Huangqi Guizhi Wuwu decoction (0，20，40，and 80 μg/mL) could significantly inhibit the proliferation of MDA-MB-231 cells (P＜0.001)，with a half-maximal inhibitory concentration of 81.02 μg/mL.Wound healing assay showed that compared with the control group，80 μg/mL Huangqi Guizhi Wuwu decoction could significantly inhibit the migration ability of MDA-MB-231 cells (P＜0.05).The colony formation assay showed that the extract of Huangqi Guizhi Wuwu decoction could inhibit the colony formation ability of MDA-MB-231 cells (P＜0.05).Conclusion：Huangqi Guizhi Wuwu decoction acts on the key targets including AKT1 and TP53 through the components such as quercetin and kaempferol and inhibits the proliferation，migration，and colony formation of breast cancer cells.This study reveals the antitumor effect of Huangqi Guizhi Wuwu decoction through multiple targets and pathways，which provides new strategies for the treatment of TNBC. |
| Key words: Huangqi Guizhi Wuwu decoction triple-negative breast cancer network pharmacology |
